Genetically modified non-human mammals and cells

ABSTRACT

A genetically modified non-human mammal or cell characterised in that it does not comprise a nucleic acid sequence which itself encodes any endogenous immunoglobulin heavy chain constant region locus polypeptide.

Related Applications

This application is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/GB2004/000768, filed Feb. 26, 2004, whichwas published under PCT Article 21(2) in English.

This invention relates to genetically modified non-human mammals, inparticular to genetically modified rodents such as mice, which do notencode any endogenous immunoglobulin heavy chain constant region locuspolypeptide. The invention also relates to genetically modifiednon-human cells, particularly embryonic stem cells, especially rodentcells such as mouse cells, which do not encode any endogenousimmunoglobulin heavy chain constant region locus polypeptide. Theinvention also relates to genetically modified non-human cells,particularly embryonic stem cells and genetically modified non-humanmammals produced therefrom, and their use in the production of non-humanmammals and cells from which all the endogenous immunoglobulin heavychain constant region genes (from Cμ to Cα) have been deleted from thegenome.

This invention further relates to genetically modified non-humanmammals, in particular to genetically modified rodents such as mice, inwhich the deletion of the endogenous immunoglobulin heavy chain constantregion genes has been used to allow insertion of other genes, such asexogenous immunoglobulin genes or their segments, to secure and allowimmunoglobulin heavy chain locus specific gene expression. Additionally,the invention relates to the expression of such genes produced by suchmammals, and the use of such mammals or cells thereof in the productionof modified immune systems with particular emphasis on production ofimmunoglobulins or antibodies.

Furthermore the invention relates to non-human mammals without anyendogenous constant region genes bred with compatible non-human mammalscapable of expressing exogenous immunoglobulin genes either asintroduced rearranged entities or entities in germ line configurationand on large chromosome fragments mature B-cell with further developmentin the periphery to an antibody secreting plasma cell which has switchedfrom Cμ to another C-gene, γ, ε or α, whilst maintaining its originalrearranged V(variable) region.

Brüggemann et al (PNAS 1989; 86: 6709-6723) describe production of micecarrying a human heavy chain minilocus with unrearranged Ig variable(V), diversity (D) and joining (J) elements linked to a human heavychain constant μ gene, which encodes the IgM immunoglobulin isotype. Theforeign (human) immunoglobulin genes are inserted into the germline ofthe transgenic mice, with the result that the foreign insert is presentin addition to the endogenous Ig genes. In these mice the foreign genescan rearrange to encode a repertoire of immunoglobulins of the IgMisotype.

The work by Brüggemann et al (ibid) is also described in U.S. Pat. No.5,545,807 which relates to a method of producing an immunoglobulinobtained from cells or body fluid of a transgenic animal which has hadinserted into its germline genetic material that encodes for at leastpart of an immunoglobulin of foreign origin or that can rearrange toencode a repertoire of immunoglobulins. Therein, it is suggested that“it may be convenient to use a host animal that initially does not carrygenetic material encoding immunoglobulin constant regions so that theresulting transgenic animal will use only the inserted foreign geneticmaterial when producing the immunoglobulins. This can be achieved eitherby using a naturally occurring mutant lacking the relevant geneticmaterial or, by artificially making mutants e.g. in cell linesultimately to create a host from which the relevant genetic material hasbeen removed.” However, no suggestion is made as to how to provide suchan IgC-deficient host animal and the teaching focuses only on insertionof human genetic material; no example is given in which the endogenousIg heavy chain constant region is deleted.

Targeted alterations have focussed on individual C-region genes but theremoval of Cμ, Cδ or Cε has not significantly altered progression inB-cell development [1-3]. It seems that silencing of individual C-genesor replacement [4, 5] has little effect on developmental progression dueto their functional redundancy. Similarly, even though removal of the Eμenhancer region reduced DNA rearrangement [6, 7] and replacement of theα3′ enhancer affected switching [8] these are only small perturbationsalmost negligible in immune development.

Preventing the use of H-chain C-region genes with a subsequent block inB-cell development has been achieved by two opposite approaches. Removalof the J(joining)_(H) segments eliminated D(diversity)-J rearrangement[9, 10] and the mice are devoid of Ig⁺ B-cells but continue to developB220⁺ B-cell precursors present at somewhat reduced levels

EP 0 463 151 discloses mice in which a 2.3 kb fragment of the endogenousmouse heavy chain locus, carrying the D and J1-4 genes, is removed andreplaced (by homologous recombination) with a neomycin resistance gene.The endogenous mouse IgH C genes are present in the mouse, but becausethe D and J genes are absent, rearrangement of the locus cannot takeplace and expression of the endogenous IgH C genes is blocked,preventing production of a functional message encoding an IgH C subunit.

Kitamura et al [11] (Nature 1991; 350: 423-426) describe production ofmice with a disrupted Cμ region (μMT mice). This was achieved by genetargeting, in which a 9 kb genomic fragment of Cμ and Cδ carrying a stopcodon and flanked by a 5′ neomycin resistance gene and a 3′HSV tk gene,was inserted into the membrane exons of Cμ using embryonic stem cells.The ES cells were used to generate chimaeric animals, which were bred toobtain mice heterozygous and then homozygous for the disrupted Cμregion. Mice homozygous for the disrupted Cμ transmembrane exon were Igdeficient. B-cell development is dependent on the expression of surfaceμ at the pre B-cell stage, but because expression of surface μ did notoccur in mice with the disrupted Cμ region (μMT mutation), thedevelopment of B cells was arrested at the stage of pre-B-cellmaturation and thus no mature or antibody secreting B-cells wereproduced.

In the lines with JH regions removed or with the disrupted Cμ exon someL-chain rearrangement occurs and it also appears that animals withdisrupted Cμ transmembrane exon bred to homozygosity in different mousestrains can largely overcome the block in Ig expression. This wasparticularly pronounced in the Balb/c background which revealed IgG, IgAand IgE expression in homozygous knock-out animals [12, 13] whilst inthe original C57BL/6 background only IgA was selectively expressed whichhas been interpreted as remnant of an ancient perhaps more primitiveimmune system [14].

There is a desire to produce genetically modified non-human mammals,e.g. rodents such as mice, in which the endogenous IgH genes have beensilenced, or removed, in order to produce antibodies of foreign origin,expressed from introduced genes.

A number of approaches have been used for silencing (i.e. disruption),or removal, of single endogenous Ig genes, combined with simultaneousintroduction of one exogenous human gene.

Pluschke et al (J. Immunol. Methods 1998; 215 (1-2): 27-37) used aconventional gene targeting strategy (Stief et al., J. Immunol. 1994;152: 3378) in embryonic stem cells to replace the mouse IgH constantgamma 2a (Cγ2a) gene segment with the human IgH constant gamma 1 (Cγ1);in addition to this, ES cells were generated in which the mouse IgLkappa gene segment was replaced with its human counterpart. The ES cellswere used to generate chimaeric mice.

Zou et al (Science, 1993; 262, 1271-1274) describe a Cre-loxPrecombination system that operates in mammalian cells and has been usedfor gene targeting experiments in the mouse to generate “clean”deletions of target genes in the germ line, as well as to inactivategenes in a conditional manner (based on regulated expression of Crerecombinase).

Cre is a 38 kDa recombinase protein from bacteriophage P1 which mediatesintramolecular (excisive or inversional) and intermolecular(integrative) site specific recombination between loxP sites; for areview of the system refer to Sauer in Methods of Enzymology; 1993, Vol.225, 890-900. A loxP site (the locus of crossing over) consists of two13 bp inverted repeats separated by an 8 bp asymmetric spacer region.One molecule of Cre binds per inverted repeat, or two Cre molecules lineup at one loxP site. The recombination occurs in the asymmetric spacerregion. Those 8 bases are also responsible for the directionality of thesite. Two loxP sequences in opposite orientation to each other invertthe intervening piece of DNA, two sites in direct orientation dictateexcision of the intervening DNA between the sites, leaving one loxP sitebehind. This precise removal of DNA can be used to eliminate genes (genedeletion) or to activate genes. The Cre-loxP system can also be used tointroduce genes (gene introduction). A gene flanked by two loxP sites ina direct orientation is referred to as a “floxed gene”.

Zou et al (Current Biology 1994; 4: (12) 1099-2003) describe use of theCre-loxP system in mouse embryonic stem cells to replace the mouse geneCγ1, which encodes the constant region of the heavy chain of IgG1antibodies, with the corresponding human gene Cγ1. A targeting constructwas generated in which a loxP site was cloned at the 3′end of the targetgene sequence (in this instance the mouse Cγ1) and, at a position 5′ ofthe target gene, an insertion was made of (from 5′ to 3′) a mutant geneof interest (in this instance human Cγ1), a loxP site, a negativeselection marker (HSV-tk) and a positive selection marker (neo^(r)). Inthe construct the loxP sites were in direct orientation. The targetingconstruct was introduced by transfection into ES cells, transformantswere selected on G418 by neomycin resistance. A Cre construct wasintroduced into the transformed cells to achieve transient expression ofCre. Recombination, that is excision of the sequence between the twoloxP sites (encoding HSV-tk, neo^(r) and the endogenous target genemouse Cγ1), occurred only in those cells expressing Cre recombinase. Thehuman Cγ1 sequence was situated outside the loxP sites and thus remainedinserted within the mouse genome. Negative selection using acyclovir organcyclovir was used to identify those cells in which the deletion hadtaken place, as only cells that do not express HSV-tk, i.e. those inwhich the endogenous mouse Cγ1 gene has also been deleted, were able tosurvive on those media.

Thus, Zou et al (1994) used the Cre-loxP system to introduce a human Cγ1gene and then delete a single endogenous Ig heavy chain constant regiongene, Cγ1. The exons encoding the transmembrane and cytoplasmic portionsof the IgH mouse Cγ1 were not replaced by human sequences, these wereretained to minimise the risk of disturbing membrane expression andsignalling of the humanised IgG1 in the mouse. The introduced human Cγ1gene was transmitted through the mouse germline and the resulting mutantmice were crossed with mice expressing kappa light chains with a human,instead of mouse constant region. Mice homozygous for both insertionsproduce humanised kappa chain bearing IgG1 antibodies.

Nicholson et al (J Immunol. 1999; 6888-6906) produced mice that carryYAC based human Ig heavy and both κ and λ light chain transloci in abackground in which the endogenous IgH and Igκ loci have beeninactivated. Inactivation of the IgH locus was achieved using the Cμ(μMT mutant) mice described by Kitamura (1991) supra, Igκ expression wasdisrupted by insertion of a Neo cassette in the Cκ gene (Zou et al Eur JImmunol 1995, 25, 2154).

A technical problem addressed by this invention is the production of anon-human mammal, that is unable to express any of its endogenous IgH Cgenes and thus is immunodeficient. A particular problem addressed by thepresent invention is the production of a rodent, in particular a mouse,that is unable to express any of its 8 endogenous IgH C genes. Toachieve this, it is necessary to generate a mutant non-human mammal, inparticular a rodent such as a mouse, in which the endogenous heavy chainconstant region genes are no longer present or not functionally active.

Accordingly the present invention provides a genetically modifiednon-human mammal or a genetically modified non-human mammalian cellcharacterised in that it does not comprise a nucleic acid sequence whichitself encodes any endogenous immunoglobulin heavy chain constant regionlocus polypeptide. The invention also provides a genetically modified ortransgenic mouse wherein the germ cells are free from the endogenousimmunoglobulin C gene locus. The invention further provides agenetically modified transgenic mouse or the progeny thereof, whereinthe somatic and germ cells are free from the endogenous immunoglobulin Cgene locus.

In an aspect of the invention, the genetically modified non-human mammalor cell does not comprise a nucleic acid sequence which itself encodesany immunoglobulin heavy chain constant region locus polypeptide. Inpreferred genetically modified non-human mammals and cells of theinvention, all immunoglobulin heavy chain constant (IgH C) region genesequences are absent or partially absent from the genome. Preferablyeach of the endogenous IgH C region genes is absent; more preferably theentire endogenous C region (from Cμ to Cα) is absent.

Herein, endogenous is defined as authentic, native, not foreign and notmodified by genetic engineering such as gene targeting or geneintroduction.

Genetically modified non-human mammals or cells are obtainable bytargeted deletion of all or essentially all endogenous IgH C genesequences. The deletion can be of all endogenous IgH C region genes andintervening sequences (complete exon/intron removal or clean deletion)or essentially all endogenous IgH C sequences by deletion of anextensive part of the endogenous IgH C region gene sequence such thatexpression of any of the IgH C genes is prevented. Targeted deletion canbe performed by a recombination-excision process, for example byCre-loxP recombination. Thus the invention further provides agenetically modified or transgenic non-human mammal as described hereinor a genetically modified or transgenic non-human mammalian cellpreferably an embryonic stem cell as described herein, obtainable by asite specific recombination method, preferably by a Cre-loxPrecombination method.

In site specific recombination methods for targeted deletion, a regionof nucleic acid sequence flanked by two site specific recombinationsequences is excised; following excision, a single site specificrecombination sequence remains within the genome. It is preferred thatthe site specific recombination sequence is a non-endogenous sitespecific recombination (NESSR) site. Several methods can be used toproduce a non-human mammalian cell in which the target sequence fordeletion, i.e. the endogenous IgH C locus, is flanked by NESSR sites. Inone such method, NESSR sites are sequentially integrated into the genomeof a non-human mammalian cell, preferably an embryonic stem cell, sothat a NESSR site is first introduced to a cell and integrated at oneend of the target sequence and secondly a NESSR site is introduced andintegrated at the other end of the target sequence. In an alternativemethod, the NESSR sites are introduced simultaneously to the cell forintegration at each end of the target sequence. Cells with a NESSR siteat one or other end of the target sequence can be used to producegenetically modified non-human mammals with NESSR sequences presentwithin the genome at one or other end of the target sequence. Anon-human mammal having a single NESSR site at one end of the IgH Clocus target sequence can be bred with non-human mammal having a NESSRsite at the other end of the IgH C locus target sequence, to produceprogeny with NESSR sites flanking the target sequence. Cells with NESSRsites flanking the IgH C locus target sequence can be used to producegenetically modified non-human mammals with NESSR sequences presentwithin the genome flanking the endogenous IgH C locus.

The present invention provides a genetically modified non-human mammalor cell having at least one non-endogenous site-specific recombinationsequence present within the genome downstream of, or within the lastgene of the IgH C locus and/or upstream of, or within the first gene ofthe IgH C locus. In a preferred embodiment two NESSR sites, preferablyloxP sites, are integrated within the genome downstream of, or withinthe last gene of the IgH C locus and upstream of, or within the firstgene of the IgH C locus.

An NESSR site may be present upstream at a position adjacent to thefirst gene of the IgH C locus and/or downstream at a position adjacentto the last gene of the IgH locus. By “adjacent to”, it is meant thatthe NESSR site is positioned 5′ or 3′ of the start of the first gene, orend of the last gene of the IgH C locus. This implies that the first orlast gene of the IgH C locus is the coding region nearest to the NESSR.

The invention provides a genetically modified non-human mammal, or agenetically modified non-human mammalian cell as described herein havingNESSR sites, which are preferably loxP sites, flanking the IgH C regiongenes, or inserted into the genes at each end of the IgH C region genes.

The invention provides a genetically modified non-human mammal, or agenetically modified non-human mammalian cell in which the endogenousIgH C genes are absent as described herein and a non-endogenous sitespecific recombination (NESSR) site is present within the genome,preferably the non-endogenous site specific recombination site is a loxPrecombination site.

It is preferred that the endogenous IgH C genes are deleted, but atleast part of at least one endogenous IgH C enhancer sequence isretained. This has the advantage of improving expression of foreigngenes when these are inserted at the locus and allows locus specificregulation of site-specifically introduced genes, (e.g. by usingCre-loxP insertion utilising the remaining loxP site in the deleted IgHC gene cluster). Retention of at least part of the endogenousJ-C-intronic enhancer sequence and/or at least part of the α3′ enhancersequence is particularly preferred.

In a genetically modified non-human mammal or cell of the invention, oneor more endogenous Ig H variable region, D and/or J segment nucleic acidsequences may be present. In one embodiment it is particularly preferredthat endogenous IgH variable region and D segment and J segment nucleicacid sequences are present. In an alternative embodiment an exogenousvariable region, preferably a mammalian variable region, more preferablya human variable region, is present.

A genetically modified non-human mammal or cell of the invention maycomprise one or more selectable marker(s) integrated within the genome.

A selectable marker may be positioned upstream of, or downstream of, anon-endogenous site specific recombination sequence. At least oneselectable marker may be integrated within the genome upstream of,and/or downstream of, at least one non-endogenous site specificrecombination sequence.

In a preferred embodiment, the invention provides a genetically modifiednon-human mammal, or a genetically modified non-human mammalian cell,having a selectable marker integrated upstream or downstream of thefirst and/or last endogenous IgH C gene and/or upstream or downstream ofa loxP sequence.

The selectable marker is preferably one or more of: a neomycinresistance gene; a puromycin resistance gene; a hygromycin gene or aherpes simplex virus thymidine kinase gene.

The invention further provides a genetically modified non-human mammal,or a genetically modified non-human mammalian cell as described hereincharacterised in that a different selectable marker is integrated ateach end of the IgH C region.

Thus the invention also provides a non-human mammal or non-humanmammalian cell, preferably a rodent cell, more preferably a mouse cell,most preferably a mouse embryonic stem cell, free from the endogenousimmunoglobulin heavy chain locus and comprising one or more gene(s)encoding a selectable marker.

A genetically modified non-human mammal of the invention can be rodent,murine, ovine, porcine, equine, canine, feline or the like, but ispreferably a rodent, more preferably murine, most preferably a mouse. Agenetically modified non-human mammalian cell of the invention may be anembryonic stem cell or an oocyte; and is preferably a rodent, murine,ovine, porcine, equine, canine or feline cell, or the like, preferably arodent cell, more preferably a murine cell, most preferably a mousecell. Mice are particularly preferred as their immune repertoire isextensive and they are easy to handle and breed.

The present invention provides a mouse in which all 8 endogenous heavychain constant region immunoglobulin genes (μ, δ, γ3, γ1, γ2a, γ2b, εand α) are absent, or partially absent to the extent that they arenon-functional, or in which genes δ, γ3, γ1, γ2a, γ2b and ε are absentand the flanking genes μ and α are partially absent to the extent thatthey are rendered non-functional, or in which genes μ, δ, γ3, γ1, γ2a,γ2b and ε are absent and α is partially absent to the extent that it isrendered non-functional, or in which δ, γ3, γ1, γ2a, γ2b, ε and α areabsent and μ is partially absent to the extent that it is renderednon-functional.

By partially absent it is meant that the endogenous IgH constant regiongene sequence has been deleted or disrupted to the extent that nofunctional endogenous IgH C gene product is encoded at the IgH C locus,i.e. that no functional endogenous IgH C gene product could be expressedfrom the locus.

The present invention further provides a non-human mammalian embryonicstem (ES) cell characterised in that the endogenous Ig heavy chainconstant region genes are absent or partially absent. Preferably all ofthe endogenous IgH C region genes are absent; more preferably all theknown endogenous IgH C genes are absent.

In a preferred embodiment the ES cell is a deletion mutant mouseembryonic stem cell in which all 8 endogenous heavy chain constantregion immunoglobulin genes μ, δ, γ3, γ1, γ2a, γ2b, ε and α are absentor partially absent to the extent that they are non-functional, or inwhich genes δ, γ3, γ1, γ2a, γ2b and ε are absent and the flanking genesμ and α are partially absent to the extent that they are renderednon-functional, or in which genes μ, δ, γ3, γ1, γ2a, γ2b and ε areabsent and α is partially absent to the extent that it is renderednon-functional, or in which genes δ, γ3, γ1, γ2a, γ2b, ε and α areabsent and μ is partially absent to the extent that it is renderednon-functional.

The deletion mutant non-human mammal, preferably a rodent, morepreferably a mouse, can be bred with a compatible non-human mammal thatis able to express one or more functionally active IgH C genes,preferably one or more functionally active human IgH C genes, e.g. adeletion mutant mouse can be bred with a mouse capable of expressing oneor more functionally active human IgH C genes. The heterozygous progeny(F1) of this cross can be inter-bred to produce heterozygous andhomozygous progeny (F2) of a non-human mammal, preferably a mouse, thatis not able to express the endogenous IgH C genes and instead is able toexpress only foreign, preferably human, IgH C gene(s).

As shown in other Ig knock-out mouse strains expressing Ig transgenes,the presence of mouse C genes can result in the production of chimerichuman (i.e. foreign)—mouse Ig chains by trans-switching ortrans-splicing mechanisms that bring gene segments on differentchromosomal locations together (reviewed in Brüggemann and Taussig,Curr. Opin. Biotechn., 8, 455-458, 1997). Thus, an advantage of havingdeleted the entire or essentially the entire endogenous IgH C generegion is that in F2 progeny, having and expressing an introduced, e.g.exogenous IgH gene or locus, the endogenous IgH C gene locus cannot bere-activated to produce unconventional switch or splice products.Accordingly, an advantage of producing a non-human animal with silencedendogenous Ig genes and introduced human Ig genes is that no mixedmolecules (e.g. mouse IgH and human IgL) can be produced and thusimmunisation of that animal allows the production of specific fullyhuman antibodies.

The invention provides a genetically modified non-human mammal derivedfrom a genetically modified non-human mammal as described herein, orfrom a genetically modified non-human cell as described herein, andprovides a genetically modified non-human cell derived from agenetically modified non-human mammal as described herein.

The invention provides a method for producing a genetically modifiednon-human cell comprising:

-   -   (a) (i) transfecting a non-human cell with a targeting construct        for integration upstream of, or within the first IgH C gene of        the IgH C locus, said targeting construct comprising a        non-endogenous site specific recombination sequence and a        selectable marker, selecting for a cell in which the selectable        marker is present and screening said cell for integration of the        recombination sequence, and,        -   (ii) transfecting a cell produced in (a)(i) with a targeting            construct for integration downstream of, or within the last            IgH C gene of the IgH C locus, said targeting construct            comprising a selectable marker and a non-endogenous            site-specific recombination sequence, selecting for a cell            in which the selectable marker is present and screening said            cell for integration of the recombination sequence; or,    -   (b) (i) transfecting a non-human cell with a targeting construct        for integration downstream of, or within the last IgH C gene of        the IgH C locus, said targeting construct comprising a        non-endogenous site-specific recombination sequence and a        selectable marker, selecting for a cell in which the selectable        marker is present, and screening said cell for integration of        the recombination sequence, and,        -   (ii) transfecting a cell produced in (b)(i) with a targeting            construct for integration upstream of, or within the first            IgH C gene of the IgH C locus, said targeting construct            comprising a non-endogenous site-specific recombination            sequence and a selectable marker, selecting for a cell in            which the selectable marker is present, and screening said            cell for integration of the recombination sequence; or,    -   (c) co-transfecting a non-human cell with a targeting construct        for integration upstream of, or within the first IgH C gene of        the IgH C locus and with a targeting construct for integration        downstream of, or within the last IgH C gene of the IgH C locus,        each of said targeting constructs comprising a non-endogenous        site specific recombination sequence and each having a        selectable marker, selecting for a cell in which the selectable        marker(s) is/are present, and screening said cell for        integration of the recombination sequence; and optionally,    -   (d) providing to a cell obtained in (a)(ii), (b)(ii) or (c) a        recombinase active at the non-endogenous site-specific        recombination sequence and screening for deletion events.

The recombinase in optional step (d) can be provided by an expressionvector, i.e. by introduction of an expression vector into the cell. In apreferred method, the non-endogenous site-specific recombinationsequence is a loxP site and in optional step (d), the recombinase is aCre recombinase.

It is preferred that the genetically modified non-human cell is anembryonic stem cell or an oocyte. The genetically modified non-humancell can be a rodent cell, more preferably a mouse cell.

Any suitable cloning vector may be used to generate the targetingconstruct, cloning strategies are described by Sambrook, Fritsch andManiatis in Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989. Desirably, the targeting construct may carry oneor more marker genes; suitable markers are known, especially suitableare those that allow for positive selection. Of particular interest isthe use of the gene for neomycin phosphotransferase (“neo”), whichconfers resistance to G418, also suitable is the puromycin resistancegene (“puro”) or the hygromycin resistance gene; neomycin and/orpuromycin resistance genes are preferred.

In the targeting construct, upstream and/or downstream from the targetgene, may be a gene which provides for identification of whether ahomologous double crossover has occurred (negative selection). TheHerpes simplex virus thymidine kinase gene (HSV-tk) may be used as anegative selection marker, since cells producing thymidine kinase may bekilled by acyclovir or gancyclovir.

Once a targeting construct has been prepared and any undesirablesequences removed, the construct can be introduced into the target cell,for example an ES cell or an oocyte. Any convenient technique forintroducing the DNA into the target cell may be employed. Forconventional gene targeting (usually constructs up to 20 kb), DNA ismost frequently introduced by electroporation (see Zou et al., Eur. J.Immunol., 25, 2154-62, 1995) whilst for secondary modifications, such asCre-loxP mediated integration, electroporation can be used forintegration of smaller constructs and other methods such as lipofectionand yeast spheroplast/cell fusion for YACs (yeast artificialchromosomes) and calcium phosphate-mediated DNA transfer forchromosome-fragments or mammalian artificial chromosomes which wouldallow integration of several 100 kb up to the Mb range. Thus,electroporation is the preferred technique for introduction of small DNAfragments (up to 50 kb) into the target cell, the other methods listedare suitable and perhaps advantageous for the introduction of larger DNAsequences (>50 kb).

After transformation or transfection of the target cells, they may beselected by means of positive and/or negative markers. As previouslyindicated, positive markers such as neomycin and/or puromycin resistancegenes can be used. Those cells with the desired phenotype may then befurther analysed by restriction analysis, electrophoresis, Southern blotanalysis, PCR, or the like.

PCR may also be used to detect the presence of homologous recombination.PCR primers can be used that are complementary to a sequence within thetargeting construct, and complementary to a sequence outside theconstruct and at the target locus. DNA molecules are obtained in the PCRreaction only when both the primers are able to bind to thecomplementary sequences, i.e. only if homologous recombination hasoccurred. Demonstration of the expected size fragments, verified bysequencing, supports the conclusion that homologous recombination hasoccurred.

While the presence of the marker gene in the genome indicates thatintegration has taken place, it is necessary to determine whetherhomologous integration has occurred. Methods for achieving this areknown in the art, such as using DNA analysis by Southern blothybridisation to establish the location of the integration. By employingprobes for both the insert and the sequences at the 5′ and 3′ regionsdistant to the flanking region where homologous integration would occur,it can be shown that homologous targeting has been achieved. Anadvantage is that external probes adjacent to the targeting DNA andnewly introduced restriction sites, for example by a selectable markergene, can be used for identification of the targeted alteration

Thus screening, preferably by PCR, can be used to confirm integration ofNESSR sites such as loxP sites in the correct position, on the sameallele and in the correct (direct) orientation to allow gene removal bydeletion. Screening using methods such as PCR can also be used to detectdeletion events.

An embryonic stem cell as described herein, e.g. obtainable by the abovemethod, can be used for the production of a genetically modifiednon-human mammal.

The above-described processes may be performed first to inactivate theconstant heavy chain loci in an embryonic stem cell, the cells may thenbe injected into a host blastocysts and developed into a chimaericanimal. Suitable methods are described, for example, in Hogan et al,(Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994).Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring HarbourPress NY). Chimaeric animals are bred to obtain heterozygous hosts.Then, by breeding of the heterozygous hosts, a homozygous host may beobtained.

Accordingly, the invention provides a method for producing a geneticallymodified non-human mammal characterised in that an embryonic stem cellas described herein is introduced into a host blastocyst and developedinto a chimaeric animal.

This can be achieved by a method characterised by:

-   (a) introducing a non-human mammal embryonic stem cell as described    herein into a compatible non-human mammal blastocyst, and-   (b) transplanting the blastocyst obtained in (a) into a compatible    non-human mammalian foster mother to obtain a chimaeric non-human    mammal, and optionally, screening for the selectable marker(s),    and/or non-endogenous site specific recombination sequence(s),    and/or for deletion of all or essentially all endogenous IgH C gene    sequences.

A chimaeric non-human mammal produced by these methods can be bred toobtain heterozygous progeny. The heterozygous progeny can be inter-bredto obtain homozygous progeny.

The present invention also provides a method for producing a geneticallymodified non-human mammal according to the invention comprising:

-   (a) injecting a non-human mammalian ES cell clone having two    integrated loxP sites as described herein into a non-human mammalian    blastocyst,-   (b) transplanting the blastocyst into a compatible non-human    mammalian foster mother to obtain a progeny chimaeric non-human    mammal,-   (c) optionally screening for loxP,-   (d) breeding the progeny to obtain a non-human mammal having two    integrated loxP sites on the same allele,-   (e) cross-breeding a non-human mammal having two integrated loxP    sites with a compatible Cre expressing non-human mammal,-   (f) screening the progeny for deletion mutants, preferably by PCR,    or-   (g) generating genetically modified non-human mammals, e.g. mice,    with two integrated loxP sites on one allele, or on one locus or on    2 separate alleles or loci, by cross breeding of mice derived either    by transgenesis or the ES cell route,-   (h) inter- or intra-allelic locus deletion upon Cre expression.

The invention also provides a method for producing a geneticallymodified non-human mammal characterised by cross-breeding a geneticallymodified non-human mammal homozygous for integration of a non-endogenoussite-specific recombination sequence upstream of, or within the firstIgH C gene of the IgH C locus with a compatible genetically modifiednon-human mammal homozygous for integration of a non-endogenoussite-specific recombination sequence downstream, or within the last IgHC gene of the IgH C locus, to obtain heterozygous progeny and optionallyinterbreeding the heterozygous progeny to obtain progeny homozygous forboth integrations.

The progeny homozygous for both integrations can be cross-bred with acompatible non-human mammal capable of expressing a recombinase activeat the non-endogenous site specific recombination sequence to obtainprogeny; with IgH C gene deletion, which optionally can be screenedusing suitable methods to detect IgH C gene deletion.

The non-endogenous site specific recombination sequence(s) can be loxPsites and the recombinase a Cre recombinase.

Progeny heterozygous or homozygous for loxP at both loci can becross-bred with a compatible non-human mammal capable of expressing Crerecombinase to obtain a progeny non-human mammal that does not comprisea nucleic acid sequence which itself encodes any endogenous Ig heavychain constant region locus polypeptide on one or both alleles.

A transgenic non-human mammal capable of expressing Cre recombinase maybe prepared by microinjection of linearised Cre plasmid into malepronucleus of F1 non-human mammal embryos to produce Cre expressingnon-human mammal strain.

Using methods of the invention described herein a genetically modifiednon-human mammal can be obtained that does not comprise a nucleic acidsequence which itself encodes any endogenous Ig heavy chain constantregion polypeptide.

The invention provides a method for producing a genetically modifiednon-human mammal capable of expressing one or more exogenous genes,characterised by breeding a genetically modified non-human mammal thatdoes not comprise a nucleic acid sequence which itself encodes anyendogenous immunoglobulin heavy chain constant region locus polypeptide,with a compatible non-human mammal that encodes and is capable ofexpressing one or more exogenous gene(s), to obtain progeny heterozygousfor the one or more exogenous gene(s), and optionally inter-breeding theheterozygous progeny to produce progeny homozygous for the one or moreexogenous gene(s).

An exogenous gene is a gene which is foreign, i.e. non-native, to thehost non-human mammal or cell.

Thus the invention may be used to produce a non-human mammal, that ispreferably a rodent, more preferably a mouse, that is capable ofexpressing foreign, preferably human immunoglobulin gene(s), by breedingthe genetically modified non-human mammal, as defined herein that isunable to express functionally active (endogenous) IgH C genes, with acompatible non-human mammal, preferably a rodent, more preferably amouse, that is able to express one or more functionally active foreign,preferably human IgH C genes. This enables inter-species gene/locusexchange to produce selected progeny (heterozygous or homozygous) withone or more functionally active exogenous, preferably human gene(s) ofthe desired traits in a background where the corresponding genes of thenon-human mammal are silenced or removed.

A method is provided for producing a genetically modified non-humanmammal or cell capable of expressing one or more exogenous gene(s)comprising introduction of one or more exogenous gene(s) into anon-human mammalian cell as described herein that does not comprise anucleic acid sequence which itself encodes any endogenous immunoglobulinheavy chain constant region polypeptide. It is preferred that thenon-human mammalian cell is an embryonic stem cell or an oocyte. Whenthe non-human mammalian cell is an ES cell, it is preferred that the oneor more exogenous gene(s) are introduced by transfection. When thenon-human mammal cell is an oocyte (egg cell) it is preferred that theone or more exogenous gene(s) are introduced by DNA micro-injection.Preferably the one or more exogenous gene(s) are inserted into thegenome of the non-human mammal or cell, most preferably the one or moreexogenous gene(s) are inserted into a non-endogenous site specificrecombination sequence.

An alternative method for producing a genetically modified non-humanmammal capable of expressing one or more exogenous gene(s) is provided,that comprises cross-breeding a non-human mammal that does not comprisea nucleic acid sequence which itself encodes any endogenousimmunoglobulin heavy chain constant region polypeptide with a compatibletransgenic mammal having one or more exogenous gene(s) associated withor flanked by a non-endogenous site specific recombination sequence andhaving a recombinase active at the non-endogenous site specificrecombination sequence to obtain progeny and optionally screening theprogeny for insertion of the one or more exogenous gene(s).

In the above methods for producing a genetically modified non-humanmammal or genetically modified non-human mammalian cell, capable ofexpressing one or more exogenous genes, it is preferred that thenon-endogenous site specific recombination sequence is a loxP sequenceand insertion is by Cre—lox P integration. The genetically modifiednon-human mammal is preferably a rodent, more preferably a mouse.

In order to provide for the production of xenogeneic (exogenous) bindingproteins, (e.g. foreign antibody proteins) in a host, it is necessarythat the host be competent to provide the necessary enzymes and otherfactors involved with the production of antibodies (e.g. the cellularrecombination machinery), while lacking the endogenous genes for theexpression of the heavy IgC sub-units of immunoglobulins and thus notable to express the remaining V, D and J segments after DNArearrangement. Thus, those enzymes and other factors associated withgerm line re-arrangement, splicing, somatic mutation, and the like arepreferably functional in the host. However, a functional natural regioncomprising the various exons associated with the production ofendogenous immunoglobulin heavy chain constant regions will beabsent/deleted in certain embodiments of the invention.

In a deletion and replacement strategy, the exogenous genetic materialfor insertion may be produced from a mammalian source, preferably ahuman source, or may be produced synthetically. The material may codefor at least part of a known immunoglobulin or may be modified to codefor at least part of an altered immunoglobulin. Suitable techniques forthese processes are well known.

In the case of the deletion and replacement strategy, where thexenogeneic DNA insert is large, complete Ig loci (1-3 Mb) could beinserted. The use of the Cre-loxP replacement strategy would then allowlocus removal and insertion of a different locus.

The exogenous gene or genes is preferably an Ig H gene or Ig H genes,more preferably an IgH C gene or IgH C genes. The exogenous gene orgenes can be a human gene or human gene(s), preferably the exogenousgene(s) are human Ig heavy chain constant region genes, more preferablya human Ig heavy chain constant region locus, or a human Ig heavy chainlocus, having V, D, J and/or C regions.

In the human, the immunoglobulin heavy chain locus is located onchromosome 14. In the 5′-3′ direction of transcription, the locuscomprises a large cluster of variable region genes (V_(H)), thediversity (D) region genes, followed by the joining (J_(H)) region genesand the constant (C_(H)) gene cluster. The size of the locus isestimated to be about 2,500 kilobases (kb). During B-cell development,discontinuous gene segments from the germ line IgH locus are juxtaposedby means of a physical rearrangement of the DNA. In order for afunctional heavy chain Ig polypeptide to be produced, threediscontinuous DNA segments, from the V_(H), D, and J_(H) regions must bejoined in a specific sequential fashion; V_(H) to DJ_(H), generating thefunctional unit V_(H)DJ_(H). Once a V_(H)DJ_(H) has been formed,specific heavy chains are produced following transcription of the Iglocus, utilising as a template the specific V_(H)DJ_(H)C_(H) unitcomprising exons and introns. There are two loci for Ig light chains,the K locus on human chromosome 2 and the λ locus on human chromosome22. The structure of the IgL loci is similar to that of the IgH locus,except that the D region is not present. Following IgH rearrangement,rearrangement of a light chain locus is similarly accomplished by V_(L)and J_(L) joining of the κ or λ chain. The sizes of the λ and κ loci areeach 1-3 Mb. Expression of rearranged IgH and an Igκ or Igλ light chainin a particular B-cell allows for the generation of antibody molecules.

The human Ig heavy chain locus V, D, J and/or C regions can be ingermline configuration, or can be productively arranged. In germlineconfiguration the exons are spaced by intervening sequences thus genesequence must be rearranged for expression of the gene(s). Whenproductively arranged, intervening sequences have been removed from thegene sequence and thus re-arrangement of gene sequence is not requiredfor expression of the gene(s).

The invention provides a non-human mammal or cell capable of expressingone or more exogenous genes, obtainable by a method described herein andprovides the use of a non-human mammal or cell in the production ofexogenous immunoglobulin, preferably human immunoglobulin.

An exogenous immunoglobulin is an immunoglobulin which is non-native tothe host mammal or cell in which it is produced; in some embodiments ofthe invention it is preferred that the exogenous immunoglobulincomprises a mammalian variable region which is non-native to the hostmammal or cell in which it is produced, more preferably the mammalianvariable region is a human variable region.

It has been found that a transgenic non-human mammal can producechimaeric or foreign immunoglobulin (derived from inserted exogenousgenetic material) in response to an immunogen subsequently introduced tothe transgenic non-human mammal. Accordingly, by introducing foreign,e.g. human, genetic material encoding for substantially the entirespecies-specific regions of an immunoglobulin it may be possible tostimulate the transgenic non-human mammal to produce foreignimmunoglobulin to any antigen introduced to the animal. The transgenicanimal could thus provide a highly useful, convenient and valuablesource of human immunoglobulins to a large range of antigens.Furthermore, there would be no interference due to endogenous IgH Cpolypeptide being simultaneously expressed.

Accordingly the present invention provides a method for production of animmunoglobulin comprising use of a non-human mammal or cell of theinvention, which is capable of expressing one or more exogenous genes,the immunoglobulin being an exogenous immunoglobulin with respect to thehost mammal or cell in which it is produced. In a preferred aspect theexogenous immunoglobulin comprises a mammalian variable region that isnon-native to the host mammal or cell in which it is produced, morepreferably the mammalian variable region is a human variable region. Inanother preferred aspect the exogenous immunoglobulin is a humanimmunoglobulin

When a non-human mammal is employed in a use or method for production ofan exogenous immunoglobulin, the non-human mammal is a preferably arodent, more preferably a mouse.

When a non-human mammalian cell is employed in a use or method forproduction of an exogenous immunoglobulin, the non-human mammalian cellis a preferably a rodent cell, more preferably a mouse cell.

The present invention also provides an immunoglobulin obtainable orobtained by a use or method of the invention for the production of anexogenous immunoglobulin.

The present invention also provides a human immunoglobulin obtainable orobtained by a use or method of the invention for the production of anexogenous immunoglobulin.

Further provided is an immunoglobulin for use as a medicament, saidimmunoglobulin being obtainable or obtained by a use or method of theinvention for the production of an exogenous immunoglobulin. Yet furtherprovided is the use of an immunoglobulin in the manufacture of amedicament, said immunoglobulin being obtainable or obtained by a use ormethod of the invention for the production of an exogenousimmunoglobulin. Also provided is a medicament composition comprising animmunoglobulin according to the invention and a pharmaceuticallyacceptable excipient.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the strategy for heavy chain immunoglobulin constantregion gene removal. A loxP sequence upstream of a selectable markergene (puromycin) was inserted at the most 5′C gene and anotherselectable marker gene (neomycin) upstream of a loxP sequence wasinserted downstream of the last and most 3′Cα gene at the 3′ enhancer.Upon Cre expression, this strategy resulted in the removal of a ˜200 kbregion with all C genes.

FIG. 2 illustrates the targeting construct for the 3′ region,restriction digests confirming correct assembly are provided on the lefthand side.

FIG. 3 illustrates a test Southern blot showing the germline (GL)fragments when hybridising with the 3′ ext(ernal) probe indicated.Samples with homologous integration or knock-out (KO) produce anadditional ˜10 kb SacI and ˜4 kb BamHI band in addition to a weakergermline band for the unmodified allele.

FIGS. 4 and 5 illustrate that following transfection of ZX3 (ZX3 is anin house designation for murine embryonic stem cells produced usingmethods described by Hogan, B., R. Beddington, F. Costantini, and E.Lacy. 1994 in Manipulating the mouse embryo, a laboratory manual. ColdSpring Harbor Laboratory Press), embryonic stem cells were selected andscreened and from 601 ES cell clones, one targeting event, clone no.355, was identified. Further Southern blot analysis of clone 355 isshown in FIG. 5 left hand side.

FIG. 6 illustrates the Cμ targeting construct and the sequence analysisof loxP in the 2 targeting constructs, which verified their correctorientation to allow Cre-mediated deletional removal of all C genes.

FIG. 7 shows the scheme for targeted integration and deletion of the Cgene cluster.

FIG. 8 shows Southern blot analysis of potential 355 (3′E) homozygousderived from ES clone 355 and their crossing to homozygosity. The ˜4 kbband indicates the targeting event, the ˜6.5 kb band is the germlineband and ++ indicates targeted integration on both alleles.

FIG. 9 shows the 355 ES cell clone that was used for further targetingwith the Cμ targeting vector. External probes are indicated as A and B.This resulted in one dual targeting event, clone 212.

FIG. 10 shows how integration was verified. Clone 212 carries twotargeted integrations, upstream and downstream of the C gene cluster.Transfection of the ES cells with a Cre vector allowed deletion analysisand suggested that both targeting events were on one allele.

FIG. 11 shows that clones after Cre transfection appear to produce a PCRband using primers 1 and 4 illustrated in FIG. 7. However, one bandindicating targeted integration, primers 1 and 2, remained. This mayindicate mixed clones or that the Cre-loxP mediated removal is notachieved in all cells of the clones.

FIG. 12 shows PCR analysis of potential germline transmission mice(selected by coat colour) for both targeted integrations 3′E and μ.Unexpectedly, about half the number of germline transmission mice had anallele carrying either the 5′ or 3′ targeting event, but not bothevents. Of 19 mice, 6 were negative, 7 positive for targeted integrationin 3′E, 5 positive for targeted integration in μ and one was positivefor both targeting events. As germline transmission was about 50% (halfof the mice with the correct coat colour did not have the targetmodification) it seems that the two targeted integrations are on oneallele but that cross-over frequently separated the two regions.

FIG. 13 shows the results of PCR analysis of a larger number ofpotential germline transmission mice (selected on the basis of coatcolour) which resulted in identification of 7 mice that carry bothtargeting events 3′E and μ. Of 51 mice, 22 were negative, 14 positivefor targeted integration in 3′E, 7 positive for targeted integration inμ and 7 were positive for both 3′E and μ targeting events.

FIG. 14 shows the scheme for PCR analysis of IgHC knock out (deletion)mice.

FIG. 15 shows the results of PCR analysis of first and second generationoffspring obtained following crossing the two germline transmission micecarrying both targeting events, μ^(lox) and 3′E^(lox), with Cre⁺animals.

FIG. 16 shows the results of flow cytometry analysis of bone marrow andspleen cells from homozygous CΔ (IgHC deletion) mice.

FIG. 17 shows (A) the results of analysis of D-J and V-D-J rearrangementby PCR, performed to test the engagement of the IgH locus inrearrangement of DNA from bone marrow cells, and (B) single-step RT-PCRtranscriptional analyses of RNA prepared from bone marrow cells andsingle-step RT-PCR.

FIG. 18 shows the results from ELISA performed on serum from F1, CΔ andμMT mice; in the serum of CΔ mice, no presence of any Ig or individualfractions of IgM, IgG or IgA was found.

EXAMPLES Example 1 Preparation of the Targeting Construct for the 5═ CμRegion

A λ phage library, obtained from E14 ES (embryonic stem) cell DNA(Sambrook, Frisch, Maniatis, Molecular Cloning, A laboratory manual,Cold Spring Harbor Laboratory Press, 1989), was hybridised with a 4.5 kbBamHI fragment comprising Cμ (Zou et al, Int. Immunol. 13, 1489-1499,2001) several positive clones were identified and mapped. Hybridisationmethods are well documented in the literature and known by researchersskilled in the art.

A loxP site was added to the puromycin gene (Tucker et al., Genes Dev.,10, 1008-1020, 1996) by PCR (forward primer oligo BamHI-loxP-puro:5′TTTGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATCGACCT CGAAATTCTACCGGG3′(SEQ ID NO: 1) and reverse primer oligo BclI-puro: 5′TTTGATCAGCTGATCTCGTTCTTCAGGC 3′ (SEQ ID NO: 2) which allowed theretrieval of loxP-puro on a BamHI-BclI fragment).

The ˜6.5 kb fragment from JH3/4 to Cμ1 and the ˜5.5 kb fragment from Cμ2to Cδ were linked with a loxP-puromycin resistance gene on a ˜2 kbBamHI-BclI fragment (see FIG. 6).

The targeting construct for Cμ (μ^(lox)) was assembled by subcloning ofBamHI-BglII fragments into pUC19 (Invitrogen). The 3′ EcoRI site wasreplaced by NotI using partial digest and blunt end linker insertion. Inthe resulting 5′ targeting construct for Cμ, a loxP sequence wasinserted upstream of a selectable marker gene (puromycin) which wasinserted at the Cμ gene.

Example 2 Preparation of the Targeting Construct for the α3′ Region

The λ phage library (see above), obtained from E14 ES (embryonic stem)cell DNA was hybridised with a 3.5 kb BglII fragment comprising the ratα3′enhancer (Pettersson et al, Nature, 344, 165-168, 1990) and severalpositive clones were identified and mapped.

The α3′enhancer region on a ˜9 kb SacI fragment was cloned into pUC19and the internal EcoRV site was changed to SpeI by partial digest andblunt end linker insertion. This unique site allowed the integration ofa ˜1.3 kb Neomycin-loxP gene on a compatible NheI fragment (see FIG. 2).A loxP site was added to the neomycin resistance gene (Stratagene, LaJolla, Calif.) by blunt end insertion of loxP from pGEM-30 (Gu, H.,Y.-R. Zou, and K. Rajewsky. Cell, 73, 1155-1164, 1993) and byoligonucleotide insertion (Sauer, Mol. Cell. Biol., 7, 2087-2096, 1987)in the α3′ targeting construct (3′E^(lox)) downstream of the neomycingene.

Example 3 Confirming Orientation of the loxP Sites

Correct orientation of the loxP site in each targeting construct wasverified by DNA sequencing (see FIG. 6). The loxP sites must be in thesame linear orientation to each other so that upon targeted insertion ofboth loxP sites Cre-mediated deletional removal can be obtained. Here,this allows the removal of both inserted selectable marker genes and theregion between Cμ and the 3′α enhancer at the end of the C gene cluster.

Example 4 Preparation of ES Cells

ZX3 ES cells, obtained from the 129 sv mouse strain were produced usingmethods described by Hogan, B., R. Beddington, F. Costantini, and E.Lacy. 1994 in Manipulating the mouse embryo, a laboratory manual. ColdSpring Harbor Laboratory Press. ZX3 is an in house designation for thesemurine embryonic stem cells derived by Zou Xiangang upon his 3^(rd)attempt.

Examples 5 ES Cell Transfection and Southern Hybridisation

The Cμ targeting construct was linearised using BamHI and NotI and theα3′enhancer targeting construct was linearised with BglII and EcoRV (seeFIG. 3). For each construct separately, about 10 μg purified fragment(purification kit #28304, Qiagen, Crawley, West Sussex, UK) was mixedwith ˜10⁷ ZX3 ES cells, obtained from the 129 sv mouse strain, andsubjected to electroporation and selection as described (Zou et al, Eur.J. Immunol., 25, 2154-2162, 1995; Zou et al, Int. Immunol. 13,1489-1499, 2001). DNA from G418 resistant (Neo^(r)) clones for theα3′enhancer construct was prepared and analysed in Southern blots asdescribed (Sambrook, Fritsch, Maniatis, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989). This allowed theidentification of one correctly targeted clone (355) to be used forintroduction of the Cμ construct and puromycin selection which resultedin one correctly targeted clone (212) identified with correct insertioninto Cμ and the 3′αenhancer.

Hybridisation probes were a ˜0.4 kb 3′ external EcoRV-SacI fragment forthe α3′enhancer (see FIG. 5) and 5′ and 3′ external probes for Cμdescribed in Zou et al., Int. Immunol. 13, 1489-1499, 2001 (see FIGS. 9and 10). These were the 5′ external probe, a ˜0.5 kb BamHI-BglIIfragment, and a 335 bp 3′ external probe, obtained by PCR using plasmidDNA with the following oligonucleotides: forward primer5′AACCTGACATGTTCCTCC3′ (SEQ ID NO: 3) and reverse primer5′GGGATTAGCTGAGTGTGG3′ (SEQ ID NO: 4). PCR conditions were 95° C. 1 min,58° C. 1 min and 72° C. 30 sec for 30 cycles.

Southern blots were carried out as above with the results given in FIGS.3, 4 and 5 which shows the germline (GL) fragments when hybridising withthe 3′ ext(ernal) probe indicated. Samples with homologous integrationor knock-out (KO) produced an additional ˜10 kb SacI and ˜4 kb (3.8 kb)BamHI band in addition to a weaker germline band for the unmodifiedallele.

FIG. 9 shows results from ES cell clone 355 which was used for furtherintegration of the Cμ targeting vector. Southern blot analysis wascarried out using probe B and probe A (not shown) which identified onedouble targeting event, clone 212.

For Cre-mediated deletion in transient transfection 10 μg of supercoiledplasmid pBS 185 (Gibco, Cat. No. 10347-011) was mixed with 10⁷ ES cellsin 0.8 ml ES cell medium and kept at RT for 10 min. After transfer intoa 0.4 cm electrode gap cuvette (Bio-Rad, 65-2088) electric pulses wereapplied twice at 230V and 500 μF. Cells were cultured at 37° C. for 30min and then transferred at low density into 6-well plates containingfeeder cells. After 8 to 10 days in culture clones were picked andequally split in order to test their survival in selective medium. About10% of the clones perished and in their separate fractions deletion wasidentified by PCR and Southern analysis.

FIG. 10 shows how integration was verified by Southern blot analysis asdescribed above. Clone 212 carries two targeted integrations, upstreamand downstream of the C gene cluster. Transfection of the ES cells withthe Cre vector allowed deletion which increased the resultinghybridisation band as expected.

The removal of all 8 C-region genes on a ˜200 kb region involvedtargeted integration of loxP-flanked homology constructs (μ^(lox) and3′E^(lox)) into Cμ exons 1 and 2, with the removal of a 330 bpBglII-BamHI fragment, and into the 3′ enhancer, located ˜15 kbdownstream of Cα (FIG. 7). Homologous integration was identified bySouthern hybridisation using external probes (FIG. 10). This produced ina BglII digest and hybridisation with the 5′J_(H) probe an 8.3 kbgermline and a 7.8 kb targeting band, whilst the 3′E probe identified an11.2 kb germline and a 12.5 kb targeting band. Using the 3′E probe in aBamHI digest identified a 6.5 kb germline and a 4 kb targeting band. Toassess the efficiency of Cre-loxP-mediated C-gene removal Cre plasmidpBS185 was transfected and transiently expressed in double-targeted EScells. Upon C-gene deletion a 15 kb BglII fragment was obtained whenhybridising with the 5′J_(H) and, separately, with the 3′E probe and ina BamHI digest an 11.5 kb fragment was obtained with the 3′E probe.These findings agreed with the map of the locus. Analysis of individualcolonies by Southern blotting and PCR, with examples given in FIG. 10,showed locus deletion in about 10% of the clones. This agreed well withthe initial assessment of cellular up-take and expression of theCre-gene which was verified by transferring part of each colony intoselective medium where about 1 in 10 clones died. The double targeted EScell clone 212 was used to derive mice as in vitro deletion was easilyobtained which suggested that both modifications integrated on the sameallele and that in vivo deletion could possibly be achieved by breedingof germline transmission mice with Cre expressers [15].

Verification of the two targeted integration events upstream (5′ Cμ) anddownstream (α3′) of the C gene cluster was confirmed in clone 212 bytransient transfection of the ES cells with the Cre vector and deletionanalysis by PCR using primer pair 1-4 (see FIGS. 7 and 14, for P1 and P4see Examples) which showed deletion of the C gene cluster and suggestedthat targeted integration of the two constructs happened on one allele(see FIG. 11).

FIG. 11 shows that clones after Cre transfection appear to produce a PCRband using primers 1 and 4 illustrated in FIGS. 7 and 14, and in Example8. However, one band indicating targeted integration, primers 1 and 2,remained. This may indicate mixed clones or that the Cre-loxP mediatedremoval is not achieved in all cells of the clone. Note, as this is notseen in Southern blot hybridisations, FIG. 10, is it likely to representlow level contamination picked up by very efficient PCR amplification.

Example 6 Generation of Mice and Breeding

ES cell clones with targeting events (355 for the single α3′ enhancertargeting event, integration of 3^(′)E^(lox) into the α3^(′)enhancer,and 212 for the α3′enhancer (3′E^(lox)) and 5′ Cμ (μ^(lox)) targetingevents) were injected into BALB/c blastocysts, transplanted into(C57BL/J6×CBA)F1 foster mothers and chimaeric mice and germlinetransmission was obtained as described (Hogan, B., R. Beddington, F.Costantini, and E. Lacy. 1994. Manipulating the mouse embryo, alaboratory manual. Cold Spring Harbor Laboratory Press). The mice werefurther analysed by PCR as described below and were derived, bred andinvestigated in accordance with UK Home Office project licenceregulations.

Crossing animals carrying both targeting events with Cre mice (formethods see reference 15, Zou et al (2003)) resulted in locus deletion.

Examples 7 Southern Blot Analysis of Germline Transmission Mice Derivedfrom ES Clone 355

Southern blot analysis of germline transmission mice derived from ESclone 355 with the mice derived therefrom crossed to homozygosity wascarried out using the 3′ external E probe (C in FIG. 7). The ˜4 kb bandindicates the targeting event, the ˜6.5 kb band is the germline band and++ indicates targeted integration on both alleles. The results of theSouthern blot are provided in FIG. 8.

For the derivation of transgenic mice expressing Cre-proteinubiquitously, the Cre plasmid pBS185 (GibcoBRL, Life Technologies,Paisley, UK) was linearised with ScaI and purified using a DNApurification kit (#28304, Qiagen, Crawley, West Sussex, UK). DNA wasmicroinjected into the male pronucleus of F1 embryos (CBA×C57B1/6)according to standard methods (Hogan, see above) and several founderswere produced, two of which showed a high gene/locus deletion rate whencrossed with loxP mice.

FIG. 12 shows that, unexpectedly, about half the number of germlinetransmission mice obtained from clone 212 had one allele carrying eitherthe 5′ or 3′ targeting event, but not both events. As germlinetransmission was about 50% (half of the mice with the correct coatcolour did not have the site-specific modification) it seems that thetwo targeted integrations are on one allele but that cross-overfrequently separated the two regions.

Analysis of a larger number of mice analysed by PCR identified 7 micewhich carried both targeting events (FIG. 13). Breeding of these micewith Cre expressers showed locus deletion, resulting in production of amouse with deletion of the IgH C gene cluster on one allele.

Example 8 PCR Screening for IgH C Knock Out (Deletion) Mice

Primers

NAME NAME LENGTH SEQUENCE (5′ to 3′) (J. Coadwell) (L. Ren)

-   P1 V00818f.pri MIgHKO1F 27 bp AGAGCCCCCTGTCTGATAAGAATCRGG-   P1 corresponds to SEQ ID NO: 5, this is a forward primer that binds    to the μ region.-   P2 Puromycinr.pri MIgHKO2R 23 bp TGGATGTGGAATGTGTGCGAGGC-   P2 corresponds to SEQ ID NO: 6, this is a reverse primer that binds    to the μ region.-   P3 Neomycinf.pri MIgHKO3F 23 bp TGCTTTACGGTATCGCCGCTCCC-   P3 corresponds to SEQ ID NO: 7, this is a forward primer that binds    to the 3′ enhancer region.-   P4 X96607r.pri MIgHKO4R 22 bp GAGTCCCCATCCCCAAGGCTGG-   P4 corresponds to SEQ ID NO: 8, this is a reverse primer that binds    to the 3′ enhancer region.    PCR Methods

For each of the PCRs used for the mouse IgH knock-out screening thereactions were set up as follows:

Per 20 μl reaction:

-   10× buffer 2.0 μl-   2 mM dNTPs 2.0 μl-   20 mM forward primer 0.8 μl-   20 mM reverse primer 0.8 μl-   lab-prep. Taq 0.1 μl-   diluted tail DNA 1.0 μl-   water 13.3 μl    The forward and reverse primer pairs used for each PCR type were:-   i. 212 PCR MIgHKO1F (SEQ ID NO: 5) and MIgHKO2R (SEQ ID NO: 6)-   ii. 355 PCR MIgHKO3F (SEQ ID NO: 7) and MIgHKO4R (SEQ ID NO: 8)-   iii. deletion PCR MIgHKO1F (SEQ ID NO: 5) and MIgHKO4R (SEQ ID NO:    8)    The 10× buffer used was:-   500 mM KCl-   0.05% Tween20-   100 mM Tris-Cl pH 9.0-   1.5 mM MgCl₂.    The reactions were performed as follows:

5 minutes at 95° C. for 1 cycle, 30 seconds at 94° C., then 45 secondsat the appropriate annealing temperature, followed by 1 minute at 72° C.for 30 cycles, then 10 minutes at 72° C. for 1 cycle. After which thereactions were held at 6° C. until they were analysed.

The annealing temperatures used for each reaction type were:

-   -   i. 212 PCR 62° C.    -   ii. 355 PCR 62° C.    -   iii. deletion PCR 66° C.

Example 9 Allelic Mosaicism is Transmitted Throughout the Germline

Breeding results from the first 2 germline transmission mice carryingboth targeting events, μ^(lox) and 3′E^(lox), with Cre⁺ animals (1^(st)generation) and the further breeding of C-deletion (CΔ) mice to obtainhomozygous animals without any remaining IgH C-genes (2^(nd) generation)are illustrated in table 1

TABLE 1 Transmission rate of homologous integration and locus deletion.Genotype: 1^(st) generation^(a) Genotype: 2^(nd) generation^(b)homologous number homologous number integration deletion (%) integrationdeletion (%) − + 0 (0) − +  4 (11) + +  2 (5)* + + 0 (0) + − 24 (60) + −10 (26) wildtype 14 (35) wildtype 24 (63) ^(a)Germline-transmission micecarrying both targeting events (μ^(lox) and 3

^(lox)) were crossed with heteroxzgous Cre mice. From 71 firstgeneration mice analysed by PCT, 40 were Cre⁺ and for those animals thepresence of targeted integration and locus deletion is shown.^(b)Deletion mice from the 1^(st) generation (*) were further crossedwith normal F1 mice which resulted in 38 animals analysed by PCR witheither locus deletion or, separately, the targeting events or withoutany modification.

In the 1^(st) generation mice produced, Cre transmission was somewhatbetter then the 50% expected with a large number of mice carrying thetargeting events. This implied that both targeted integrations occurredon one allele although the size and complexity of the IgH locus did notallow determiation of the chromosomal linkage by molecular means.However, in these 1^(st) generation animals CΔ was only accomplished intwo mice, which also retained the PCR bands for the targeting events(FIG. 15). Such low level of Cre-loxP-mediated deletion accompanied bylocus mosaicism was not apparent in previous breedings using the CrepBS185 mice [15]. This could be due to diminished deletion efficiency inheterozygous mice due to reduced Cre levels or alternativelyinaccessibility of the IgH locus. Breeding combinations of Cre⁺ male orfemale animals with μ^(lox) 3′E^(lox) mice did not change this outcome.Nonetheless, the findings suggested that locus deletion could beachieved but not with a high efficiency in all cells. Working on theassumption that Cre-loxP-mediated deletion can be operative early infetal development proved to be correct, as further breeding of micecarrying the targeted deletion and the 2 homologous integration eventsresulted in offspring with transmitted deletion and without anyremaining targeted integration (FIG. 15, lane 4 and 5). The frequencywith which heterozygous CΔ mice were obtained from mating of mosaicfounders was considerably lower than transmission of the targetedintegrations or indeed the number of wildtype mice, which suggests thefollowing events. A fertilised egg carries one IgH wildtype allele, onedouble-targeted IgH allele and Cre randomly integrated in the genome.Cre-mediated deletion operates disproportionately, and at the 4-cellstage only one blastomere carries the deleted allele and a wildtypeallele whilst the other 3 blastomeres carry each a double-targetedallele and a wildtype allele. This distribution pattern is maintained indevelopment and upon meiosis 4 of 8 germ cells (50%) have the wildtypeallele, 3 of 8 (37.5%) the targeted allele and 1 of 8 (12.5%) thedeletion. Although the numbers in table 1 are too small to preciselymatch this calculation they show that the majority of 2^(nd) generationmice are wildtype, that an intermediate number of mice carry thetargeted integrations and, at 11%, a close match is found for the numberof CΔ mice which strongly supports the prediction that Cre-mediatedrecombination can be disproportionate at early developmental stages.

Example 10 PCR Analysis of Deletion and Rearrangement

Homologous integration in Cμ and the α3′ enhancer was identified using 2sets of oligonucleotides (see FIG. 1): (1) Cμ forward(5′AGAGCCCCCTGTCTGATAA GAATCTGG3′ SEQ ID NO: 5) and (2) puro(5′TGGATGTGGAATGTGT GCGAGGC 3′ SEQ ID NO: 6), produced a ˜485 bpfragment; while (3) neo (5′TGCTTTACGGTATCGCCGCTCCC 3′ SEQ ID NO: 7) and(4) 3′E (5′GAGTCCCCATCCCCAAGGCTGG 3′ SEQ ID NO: 8) produced a ˜500 bpfragment. Upon C gene removal oligos (1) and (4) produced a ˜613 bpdeletion band. The presence of the Cre-transgene and the γ2a gene in theendogenous IgH locus were identified using the followingoligonucleotides: Cre for 5′GGACATGTTCAGGGATCGCCAGG3′ SEQ ID NO: 9 andCre rev 5′GATAGCTGGCTGGTGGCAGATGG3′ SEQ ID NO: 10, and γ2a for5′GGCTGGGA TGGGCATAAGGATAAAGGTC3′ SEQ ID NO: 11 and a 1 to 1 mixture ofγ2a^(a) rev 5′GTAGCTATTTCTTTCCACCCAGTTCTTC3′ SEQ ID NO: 12 and γ2a^(b)rev 5′GAAAAGACTTCCTCTTTCCCAAGTGCTC3′ SEQ ID NO: 13 which were allotypespecific. Optimal PCR conditions were 94° C. 45 sec, 60° C. (Cre, γ2a)or 62° C. (Cμ-puro, neo-3′E) or 66° C. (CΔ-3′E) 1 min or 45 sec (Cre)and 72° C. 45 sec for 30 cycles followed by 10 min at 72° C. to completethe reaction.

For the analysis of D-J and V-D-J rearrangement, DNA and RNA wasprepared from bone marrow cells using Tri Reagent (Sigma) and theOne-Step RT-PCR System (Invitrogen, life technologies). Combinations ofthe following oligonucleotides were employed: a 1:1 mixture of DF (5′GCATGTCTCAAAGCACAATG 3′ SEQ ID NO: 14) and DQ52 (5′ ACCCTGGACACAGGAAACAC3′ SEQ ID NO: 15) forward primers; a 1:1 mixture of VJ558L (5′ATGGGATGGAGCTGGATCTT 3′ SEQ ID NO: 16) and VJ558CL (5′ATGGAATGGAGCTGGGTCTT 3′ SEQ ID NO: 17) forward primers; JH1-4 reverseprimer (5′ GAGACDGTGASHRDRGTBCCTKSRCC 3′ SEQ ID NO: 18 with R=A+G,K=G+T, H=A+T+C, B=G+T+C and D=G+A+T); and lamin B1, a ubiquitouslyexpressed gene as control, lamin for 5′ GTATGAGGCGGCACTAAACTCTAA 3′ SEQID NO: 19, and a 1:1 mixture of lamin rev genomic 5′GAAGCCACTGAAGAACACAAATAG 3′ SEQ ID NO: 20 and lamin rev cDNA 5′TACGAAACTCCAAGTCCTCAGTAA 3′ SEQ ID NO: 21. PCR conditions were 35 cyclesof 92° C. 15 sec, 60° C. 30 sec and 72° C. 40 sec and RT-PCR conditionswere 30 min 50° C. and 2 min 94° C. followed by 35 cycles of 92° C. 15sec, 50° C. 30 sec and 72° C. 40 sec, followed by 10 min at 72° C. tocomplete the reaction.

Example 11 Block in Development at the pre B-I Stage

To investigate the developmental capacity of the lymphocyte populationin homozygous CΔ mice, bone marrow and spleen cells were analysed byflow cytometry (FIG. 16).

Flow Cytometry Analysis

Bone marrow and spleen cell suspensions were prepared from normal F1(C57/BL6×CBA), μMT mice (μMT^(−/−) (Cμ) homozygous mutant mice [11]) andCΔ deletion mice. Bone marrow cells were stained in four colourcombinations (see FIG. 4) with PE-conjugated anti-mouse c-kit (CD117,09995B, BD PharMingen, San Diego, Calif.), APC-conjugated anti-mouseCD45R (B220, 01129A, BD PharMingen), biotin-conjugated anti-mouse CD25(01092A, BD PharMingen), FITC-conjugated monoclonal rat anti-mouse IgM(μchain specific, 04-6811, Zymed, San Francisco, Calif.),biotin-conjugated anti-mouse CD43 (01602D, BD PharMingen),FITC-conjugated anti-mouse IgD (02214D, BD PharMingen) and/orPE-conjugated anti-mouse Igκ L-chain (559940, BD PharMingen). Spleencells were stained with APC-conjugated anti-mouse CD45R,Biotin-conjugated anti-mouse IgM (μ-chain specific, 02082D, BDPharMingen), PE-conjugated anti-mouse Igκ and FITC-conjugated anti-mouseCD21/CD35 (CR2/CR1, 553818, BD PharMingen). Reactions with Biotinconjugated antibodies were subsequently incubated with Tri-colorconjugated streptavidin (SA10006, Caltag). Cells were analyzed on aFACSVantage and CELLQuest was used for data analysis.

For cell surface staining, labeled antibodies against the pan B-cellmarker B220 (CD45R) were used in combination with antibodies thatallowed the identification of the various successive differentiationstages in B-cell development. In bone marrow (FIG. 16A) this showed thatpopulations at the progenitor and precursor stage, c-kit⁺ B220⁺ pro andpre B-cells and CD43⁺ B220⁺ pre B-cells, are maintained but that moremature B220⁺ lymphocytes are lacking. The absence of a large proportionof B220⁺ B-cells is similarly pronounced in CΔ and μMT mice withdisrupted membrane exons [16] and is established at the pre B-II stagewith the lack of CD25⁺ B-cells which normally express a pre BCR with μH-chain and surrogate L-chain. The removal of the C-gene cluster led toa complete disappearance of immature and mature B-cells expressing Ig H-and L-chain and staining for surface IgM, IgD, Igκ L-chain and CD21/35positive mature B-cells (FIG. 16B) reveals their total absence.

Example 12 Transcriptional Inactivity Despite DNA Rearrangement

Preserving normal pro B-cell levels in CΔ mice suggests that earlydifferentiation events may be maintained. To test the engagement of theIgH locus in DNA rearrangement, DNA from bone marrow cells was preparedand D-J and V-D-J rearrangement was analysed by PCR. For the regionbetween DQ52 and J_(H) a ˜1200 bp fragment indicated the germlineconfiguration whilst lower size amplification bands were the product ofsuccessful D-J_(H) and V_(H)-D-J_(H) recombination. In CΔ mice extensiveDNA rearrangement was identified for the developmentally earlier D-Jjoins as well as for V-D-J recombination (FIG. 17A). PCR fragments of˜500 bp for D-J and >300 bp for V-D-J rearrangement were predicted, withlarger bands representing amplifications from remaining distal J_(H)sstill present after recombination. The CΔ mice produced bands of theexpected size range which were similar in pattern and intensity to thosefound in normal mice and μMT mice analysed in parallel. Fortranscriptional analyses RNA was prepared from bone marrow cells andsingle-step RT-PCR using RNA from ˜10⁶ cells per reaction revealed thatlittle or no rearranged H-chain transcripts were produced in CΔ mice(FIG. 17B). A remaining low level of transcription could be indicated bythe faint bands in some of the reactions, alternatively, this mayrepresent the background due to the low stringency of the reaction orthe oligonucleotides used for amplification. However, using RNA fromnormal mice as well as μMT mice produced discernible RT-PCR bands of theexpected size range which established a marked difference after H-chainDNA rearrangement between μMT mice exhibiting fully functional H-chaintranscription and C Δ mice with transcriptionally silent H-chain. Werepeated the PCR and RT-PCR analysis with ˜10⁵ and ˜5×10⁵ CD43⁺ B220⁺bone marrow cells, respectively, sorted by flow cytometry. Distinctbands but less diverse patterns were obtained for normal and μMT mice,but again CΔ mice did not yield any RT-PCR products (data not shown).PCR amplification of lamin B1, a ubiquitously expressed gene used as acontrol, produced a genomic fragment of ˜433 bp and a cDNA product of˜215 bp, identified in all mice.

The finding that CΔ mice are transcriptionally inactive agrees withtheir lack of serum antibodies.

ELISA

Serum antibodies were captured by ELISA [33] on Falcon plates (353911,Becton Dickinson) coated with 50 μl of 10 μg/ml anti-IgM (μ-chainspecific) (Sigma M-8644), anti-IgG (γ-chain specific) (Binding SiteAU272), anti-IgA (α-chain specific) (Sigma M-8769) or anti-mouse κL-chain (Sigma K-2132). Bound antibody was identified using biotinylatedanti-Ig (1/500 dilution, Amersham RPN 1001) or anti-κ (1/200 dilution,Zymed 04-6640) followed by a 1/200 dilution of streptavidin-conjugatedhorseradish peroxidase (Amersham 1052). A₄₉₂ was measured in a TitertekMultiskan MCC/340.

In ELISA no presence of any Ig or individual fractions of IgM, IgG orIgA were found in the serum of CΔ mice (FIG. 18) whilst Ig expression inμMT mice appeared to be similar to previously reported expression levels[12-14]. The defect in Ig production, re-emphasised by the lack of Igκshows that no antibody H-chain fragments or L-chains on their own arebeing expressed in CΔ mice.

Targeted insertion of loxP sites, 5′ and 3′ of the C_(H)-gene cluster,allowed Cre-mediated removal of a ˜200 kb region which produced an Igdeficient mouse line. While in vitro deletion was accomplished with theexpected efficiency by transient Cre expression, in vivo C-gene removalby breeding with ubiquitous Cre expressers proved difficult. As aresult, and despite normal transmission of the Cre-transgene, fewanimals carried the C-gene deletion (see Table 1). Such a lack ofefficiency was not observed when ˜400 kb of the Igλ L-chain locus wereremoved by breeding with the Cre mouse line [15, 17 and ENSEMBL MouseRelease 17.30.1 (NCBI 30 assembly)] and it is unclear why differentlevels of efficiency operate on the two targeted Ig loci. In the firstgeneration of mice carrying the C-gene deletion (litters from Cre⁺ micemated with μ^(lox) 3′E^(lox) mice), analysed by PCR of tail DNA, thedeletion band was always accompanied by the presence of bands for thetargeting events. A problem with the efficiency of Cre-mediatedrecombination has been linked to its promoter and it has been shown thatthe major immediate early promoter from human cytomegalovirus (CMV),used in our Cre mice, is less efficient that the equivalent murine CMVpromoter [18]. Both ubiquitous and conditional expression of theCre-gene, showed a variation in efficiency [19, 20] and this may explainwhy Cre-mediated deletion of the C-gene locus was only achievedsuccessfully in a varied proportion of cells and tissues producing amosaic pattern. Nevertheless, C-gene deletion can be accomplished earlyon to secure modified germ cell development which allowed homozygousmice without remaining C-genes to be established by breeding.

The experiments show that removal of all C-genes silences the IgH locusat the transcriptional level, but not at the DNA rearrangement stage.This implies that locus activation is fully maintained and indeed thelevels of early B-cells at the pro to pre B-cell differentiation stage,c-kit⁺ B220⁺ and CD43⁺ B220⁺, are comparable to those in normal mice.Nonetheless, with the lack of CD25⁺ B220⁺ cells (see FIG. 16), acomplete block in development is accomplished at the pre B-II stage whenrearranged Ig H-chains should be transcribed and expressed. StainingB-cell populations from bone marrow and spleen with antibodies specificfor differentiation markers revealed a very similar developmentalpattern for μMT and CΔ mice which is also confirmed at the DNA levelwhere both strains show extensive rearrangement (ref. 14 and FIG. 17).However, regarding Ig expression there are fundamental differencesbetween the CΔ and μMT strains which are not apparent using flowcytometry analysis of B-cell development where small populations ofmature B-cells can escape detection. That these mature B cells arepresent in the bone marrow of μMT mice, but not in CΔ mice, became clearin RT-PCR reactions. Here CΔ mice appear not to produce IgH transcripts,which are abundantly present in bone marrow cells from μMT mice. To ruleout any artefacts, cDNA for the identification of Ig transcripts wasproduced with internal J_(H) primers. This prevented selectiveamplification of polyadenylated RNA, an unlikely H-chain product in CΔmice lacking the 3′ untranslated region essential for mRNA processingand expression. Ig expression could be identified in the serum of μMTmice, but not in CΔ mice, which showed a complete lack of Ig in ELISA.This established that with the removal of all C-genes the IgH locusbecomes fully inoperative at the transcriptional level after DNArearrangement is completed. Unexpectedly we identified a significantamount of IgG in the serum of the μMT animals at levels that have notbeen seen in μMT mice of C57BL/6 background [14]. A reason could be thatthe μMT mice maintained as a breeding colony for a long time have lostthe true C57BL/6 background, and thus permit IgG expression [12, 13].However, the intermediate IgA levels of the μMT mice are quite similarto the levels reported for μMT in the C57BL/6 background and analysis ofindividual mice showed that wildtype levels as in μMT of BALB/cbackground [12, 13] could not be reached (data not shown).

In respect of other H-chain silencing approaches the removal of clustersof gene segments, such as all Vs, Ds, Js or C-genes, appears toguarantee locus insufficiency. This has been shown in J_(H) deletionmice where a block in B-cell development has also been reported at theCD43⁺ precursor stage [9, 21], and a lack of μ transcripts from theallele carrying the deletion was found [10]. A dysfunctional H-chainlocus silences L-chain expression and although the Igκ locus canrearrange at the same time or even earlier than the IgH locus, aproductively rearranged L-chain is only expressed upon H-chainproduction ([22] and [9] and refs therein). In J_(H) deletion mice C κrearrangement in B220⁺CD43⁺ sorted cell populations was quite similar towild type levels, whilst only a low level of germline transcripts couldbe detected in Northern blot analysis [9]. On the contrary the removalof Cμ, the first C-gene expressed and regarded essential to drive B-celldevelopment, did not produce H-chain silent mice [1]. A likely reasonfor this is that the function of one C-gene can be replaced by another.In Cμ deletion mice this was accomplished by Cδ and in Cγ2a replacement,a perhaps less important C-gene further downstream, other Cγs assumedresponsibility [4]. Similarly, the removal of Cδ was well tolerated andperhaps compensated by increased Igμ expression [2]. The assumption thatB-cell maturation after DNA rearrangement may be critically dependent onthe expression of Igμ came from transgenic mouse models, where in theinitial approaches Cμ was the only C-gene on an introduced IgH locus ingermline configuration ([23] and refs therein). However, this does notimply that other C-genes, e.g. Cγ, could not initiate similardevelopmental processes, which may be unravelled when mice with Cμ andCδ deletion are being produced. Suggestions that perhaps a moreprimitive or ancient evolutionary immune system can be operative comesfrom the observation that IgA is expressed in silenced μMT mice [14] andthat H-chain only antibodies in Camelidae do appear to bypass the Igμprecursor cell stage [24, 25]. This can be tested in the CΔ mice byCre-loxP mediated targeted gene insertion.

References

-   [1] C. Lutz, et al., IgD can largely substitute for loss of IgM    function in B cells, Nature 393 (1998) 797-801.-   [2] L. Nitschke, M. H. Kosco, G. Kohler, M. C. Lamers,    Immunoglobulin D-deficient mice can mount normal immune responses to    thymus-independent and -dependent antigens, Proc. Natl. Acad. Sci.    USA 90 (1993) 1887-1891.-   [3] G. Achatz, L. Nitschke, M. C. Lamers, Effect of transmembrane    and cytoplasmic domains of IgE on the IgE response, Science    276 (1997) 409-411.-   [4] G. Pluschke et al., Generation of chimeric monoclonal antibodies    from mice that carry human immunoglobulin Cγ1 heavy of Cκ light    chain gene segments, Immunol. Methods 215 (1998) 27-37.-   [5] Y. R. Zou, W. Müller, H. Gu, K. Rajewsky, Cre-loxP-mediated gene    replacement: a mouse strain producing humanized antibodies, Current    Biology 4 (1994) 1099-1103.-   [6] M. Serwe, F. Sablitzky, V(D)J recombination in B cells is    impaired but not blocked by targeted deletion of the immunoglobulin    heavy chain intron enhancer, EMBO J. 12 (1993) 2321-2227.-   [7] J. Chen, F. Young, A. Bottaro, V. Stewart, R. K. Smith, F. W.    Alt, Mutations of the intronic IgH enhancer and its flanking    sequences differentially affect accessibility of the JH locus,    EMBO J. 12 (1993) 4635-4645.-   [8] M. Cogne et al., A class switch control region at the 3′ end of    the immunoglobulin heavy chain locus, Cell 77 (1994) 737-747.-   [9] J. Chen et al., Immunoglobulin gene rearrangement in B cell    deficient mice generated by targeted deletion of the J_(H) locus,    Int. Immunol. 5 (1993) 647-656.-   [10] H. Gu, Y. R. Zou, K. Rajewsky, Independent control of    immunoglobulin switch recombination at individual switch regions    evidenced through Cre-loxP-mediated gene targeting, Cell 73 (1993)    1155-1164.-   [11] D. Kitamura, J. Roes, R. Kuhn, K. Rajewsky, A B cell-deficient    mouse by targeted disruption of the membrane exon of the    immunoglobulin μ chain gene, Nature 350 (1991) 423-426.-   [12] Z. Orinska et al., Novel B cell population producing functional    IgG in the absence of membrane IgM expression, Eur. J. Immunol.    32 (2002) 3472-3480.-   [13] M. Hasan, B. Polic, M. Bralic, S. Jonjic, K. Rajewsky,    Incomplete block of B cell development and immunoglobulin production    in mice carrying the μMT mutation on the BALB/c background, Eur. J.    Immunol. 32 (2002) 3463-3471.-   [14] A. J. Macpherson et al., IgA production without μ or δ chain    expression in developing B cells, Nat. Immunol. 2 (2001) 625-631.-   [15] X. Zou, T. A. Piper, J. A. Smith, N. D. Allen, J. Xian, M.    Brüggemann, Block in development at the pre-B-II to immature B cell    stage in mice without Igκ and Igλ light chain, J. Immunol.    170 (2003) 1354-1361.-   [16] T. Nikolic, G. M. Dingjan, P. J. Leenen, R. W. Hendriks, A    subfraction of B220⁺ cells in murine bone marrow and spleen does not    belong to the B cell lineage but has dendritic cell characteristics.    Eur. J. Immunol. 32 (2002) 686-692.-   [17] M. Clamp et al., Ensembl 2002: accommodating comparative    genomics, Nucleic Acids Res. 31 (2003) 38-42.-   [18] C. E. Appleby et al., A novel combination of promoter and    enhancers increases transgene expression in vascular smooth muscle    cells in vitro and coronary arteries in vivo after    adenovirus-mediated gene transfer, Gene Ther. 10 (2003) 1616-1622.-   [19] K. Sakai et al., Stage-and tissue-specific expression of a    Col2a1-Cre fusion gene in transgenic mice, Matrix Biol. 19 (2001)    761-767.-   [20] H. Guo et al., Specificity and efficiency of Cre-mediated    recombination in Emx1-Cre knock-in mice, Biochem. Biophys. Res.    Commun. 273 (2000) 661-665.-   [21] A. Jakobovits et al., Analysis of homozygous mutant chimeric    mice: deletion of the immunoglobulin heavy-chain joining region    blocks B-cell development and antibody production. Proc. Natl. Acad.    Sci. USA 90 (1993) 2551-2555.-   [22] T. I. Novobrantseva, V. M. Martin, R. Pelanda, W. Müller, K.    Rajewsky, A. Ehlich, Rearrangement and expression of immunoglobulin    light chain genes can precede heavy chain expression during normal B    cell development in mice, J. Exp. Med. 189 (1999) 75-88.-   [23] M. Brüggemann, Human monoclonal antibodies from translocus    mice. in Molecular Biology of B-cells. (2004) F. W. Alt, T.    Honjo, M. S. Neuberger (Eds.), pp 547-561.-   [24] V. K. Nguyen, A. Desmyter, S. Muyldermans, Functional    Heavy-chain Antibodies in Camelidae, Adv. Immunol. 79 (2001)    261-296.-   [25] V. K. Nguyen, X. Zou, M. Lauwereyes, L. Brys, M. Brüggemann, S.    Muyldermans, Heavy-chain only antibodies derived from dromedary are    secreted and displayed by mouse B-cells, Immunology 109 (2003)    93-101.-   [26] B. Sauer, N. Henderson, Targeted insertion of exogenous DNA    into the eukaryotic genome by the Cre recombinase, New Biol.    2 (1990) 441-449.-   [27] X. Zou, C. Ayling, J. Xian, T. A. Piper, P. J. Barker, M.    Brüggemann, Truncation of the μ heavy chain alters BCR signalling    and allows recruitment of CD5⁺ B cells, Int. Immunol. 13 (2001)    1489-1499.-   [28] S. Pettersson, G. P. Cook, M. Brüggemann, G. T. Williams, M. S.    Neuberger, A second B cell-specific enhancer 3′ of the    immunoglobulin heavy-chain locus, Nature 344 (1990) 165-168.-   [29] K. L. Tucker et al., Germ-line passage is required for    establishment of methylation and expression patterns of imprinted    but not of nonimprinted genes, Genes Dev. 10 (1996) 1008-1020.-   [30] X. Zou, J. Xian, A. V. Popov, I. R. Rosewell, M. Müller, M.    Brüggemann. Subtle differences in antibody responses and    hypermutation of λ light chains in mice with a disrupted κ constant    region, Eur. J. Immunol. 25 (1995) 2154-2162.-   [31] J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning, A    laboratory Manual (1989) Cold Spring Harbor Laboratory Press.-   [32] B. Hogan, R. Beddington, F. Costantini, E. Lacy, Manipulating    the mouse embryo, A Laboratory Manual (1994) Cold Spring Harbor    Laboratory Press.-   [33] A. V. Popov, X. Zou, J. Xian, I. C. Nicholson, M. Brüggemann, A    human immunoglobulin λ locus is similarly well expressed in mice and    humans. J. Exp. Med. 189 (1999) 1611-1619.

1. A genetically modified mouse characterised in that the completecoding region of the endogenous immunoglobulin heavy chain constantregion locus is deleted and in that one or more endogenous Ig H Variableregion, one or more endogenous Ig H D segment, and one or moreendogenous Ig H J segment nucleic acid sequences are present andcharacterised in that the genetically modified mouse does not comprise anucleic acid sequence which itself encodes any immunoglobulin heavychain constant region (IgH C) polypeptide.
 2. A genetically modifiedmouse according to claim 1, wherein all the endogenous Ig H Variableregion, D and J segment nucleic acid sequences are present.
 3. Agenetically modified mouse according to claim 1, characterised in thatit is obtainable or obtained by targeted deletion of all endogenous IgHC gene sequences.
 4. A genetically modified mouse according to claim 1characterised in that it is obtainable or obtained by Cre loxPrecombination.
 5. A genetically modified mouse according to claim 1characterised in that at least part of at least one IgH C gene enhancersequence is present.
 6. A genetically modified mouse according to claim1 characterised in that a non-endogenous site-specific recombinationsequence is present within the genome.
 7. A genetically modified mouseaccording to claim 1 characterised in that one or more selectablemarker(s) is present within the genome.
 8. A genetically modified mouseaccording to claim 6 characterised in that at least one selectablemarker is present upstream of, or downstream of, the non-endogenous sitespecific recombination sequence.
 9. A genetically modified mouseaccording to claim 7 characterised in that the selectable marker(s) isone or more selectable marker selected from a group comprising aneomycin resistance gene, a puromycin resistance gene, and a hygromycinresistance gene.
 10. A genetically modified mouse according to claim 6characterised in that the non-endogenous site-specific recombinationsequence is a loxP site.
 11. A genetically modified mouse derived from agenetically modified mouse of claim
 1. 12. A genetically modified mousecell obtained from a genetically modified mouse of claim
 1. 13. Agenetically modified mouse cell according to claim 12 characterised inthat it is an embryonic stem cell.
 14. A genetically modified mouse cellobtained from a genetically modified mouse of claim
 2. 15. A geneticallymodified mouse cell obtained from a genetically modified mouse of claim3.
 16. A genetically modified mouse cell obtained from a geneticallymodified mouse of claim
 4. 17. A genetically modified mouse cellobtained from a genetically modified mouse of claim
 5. 18. A geneticallymodified mouse cell obtained from a genetically modified mouse of claim6.
 19. A genetically modified mouse cell obtained from a geneticallymodified mouse of claim
 7. 20. A genetically modified mouse cellobtained from a genetically modified mouse of claim
 8. 21. A geneticallymodified mouse cell obtained from a genetically modified mouse of claim9.
 22. A genetically modified mouse cell obtained from a geneticallymodified mouse of claim 10.